393 research outputs found
Role of Second (p)ppGpp Synthetase MS_RHII-RSD in Mycobacterium smegmatis
No full textAdaptation to a rapidly fluctuating environment is the key to the survival of an organism. Bacteria sense and respond to stress by an overall reprogramming of the cellular processes to shut down the energy-consuming processes and switch to pathways that ensure the survival under stress. One of the strategies utilized by bacteria is to mount ‘stringent response’ which is mediated by the second messengers (p)ppGpp. (p)ppGpp governs a multitude of phenotypes in Mycobacteria and for a long time the bifunctional Rel was believed to be its only (p)ppGpp synthetase. A serendipitous detection of (p)ppGpp in a Mycobacterium smegmatis strain devoid of Rel led to the discovery of a second (p)ppGpp synthetase thereby broadening the horizon of stringent response in Mycobacteria (Murdeshwar and Chatterji, 2012). This unique protein contained a RNaseH domain along with the (p)ppGpp synthesis domain suggesting a role distinct from that of Rel. Subsequent characterisation of the protein revealed that neither domain is active in isolation raising a question about the link between these activities. Due to the crucial role played by (p)ppGpp, it becomes essential to analyse the (p)ppGpp null phenotype. Several bacterial species like Bacillus subtilis have short alarm one synthetases in addition to Rel and they have been proposed to be activated under particular stress conditions underlining the need to delineate the role of these (p)ppGpp synthetases (Nanamiya et al., 2008). Our study proposes a role for MS_RHII-RSD in vivo and deals with the phenotypic characterisation of the Δrel Δms_rhII-rsd strain.
Chapter 1 reviews the available literature in the field of stringent response and provides the rationale behind this study. The discovery of (p)ppGpp and the plethora of functions regulated by it is explained along with a description of the key players in the (p)ppGpp metabolism. The chapter stresses upon the need to investigate the significance of a second (p)ppGpp synthetase in Mycobacteria and the scope of the current study.
Chapter 2 deals with the elucidation of the in vivo significance of MS_RHII-RSD in M. smegmatis and proposes a role for the protein in R-loop removal during stress which requires both RNaseH activity and (p)ppGpp synthesis. The in vitro R-loop hydrolysis assays along with evidence for R-loop removal in M. smegmatis have been discussed along with the strategy used for the generation of the Δms_rhII-rsd strain.
Chapter 3 explores the interdependence between the RNaseH and (p)ppGpp domains in MS_RHII-RSD in an attempt to unravel the necessity of the RNase H activity in a (p)ppGpp synthetase. The generation of active-site mutants of RNaseH and RSD along with their functional and biophysical characterisation has been described in detail. Oligomerisation studies with MS_RHII-RSD revealed the importance of a hexameric form for the protein.
Chapter 4 further elaborates upon the link between the RNaseH activity and the (p)ppGpp synthesis activity and reveals a possible regulation of (p)ppGpp synthesis activity by RNA. Furthermore, the differing substrate specificities between Rel and MS_RHII-RSD are discussed. A possibility of the presence of pGpp due to MS_RHII-RSD in Mycobacteria has been outlined.
Chapter 5 describes the attempts at generating a (p)ppGpp-deficient strain of M. smegmatis and reveals the surprising presence of yet another (p)ppGpp synthetase. The generation and characterisation of the Δrel Δms_rhII-rsd strain was performed and the physiological role of MS_RHII-RSD in biofilm formation and antibiotic tolerance has been highlighted.
Chapter 6 summarizes the results of the study and points out the future directions for the work.
Appendix 1 gives a comprehensive list of strains and plasmids used in this study.
Appendix 2 provides a list of growth differences in antibiotics between the wild type and knockout strains of M. smegmatis obtained by Phenotype microarray.
Appendix 3 is a commentary on the Pup-proteasome regulation in Mycobacteria
Expanding The Horizon Of Mycobacterial Stress Response : Discovery Of A Second (P)PPGPP Synthetase In Mycobacterium Smegmatis
No full textThe stringent response is a highly conserved physiological response mounted by bacteria under stress (Ojha and Chatterji, 2001; Magnusson et al., 2005; Srivatsan and Wang, 2007; Potrykus and Cashel, 2008). Until recently, the only known players in this pathway were the (p)ppGpp synthesizing and hydrolyzing long RSH enzymes (Mittenhuber, 2001; Atkinson et al., 2011) - RelA and SpoT in Gram negative bacteria and the bifunctional Rel in Gram positive bacteria including mycobacteria. The existence of Short Alarmone Synthetases (SAS) (Lemos et al., 2007, Nanamiya et al., 2008; Das et al., 2009; Atkinson et al., 2011) and Short Alarmone Hydrolases (SAH) (Sun et al., 2010, Atkinson et al., 2011), small proteins possessing a single functional (p)ppGpp synthetase or hydrolase domain respectively, is a recent discovery that has modified this paradigm. Around the same time that the presence of the SAS proteins was reported, we chanced upon such small (p)ppGpp synthetases in the genus Mycobacterium. The stringent response in the soil saprophyte Mycobacterium smegmatis was first reported by Ojha and co-workers (Ojha et al., 2000), and the bifunctional RSH, RelMsm, responsible for mounting the stringent response in this bacterium, has been characterized in detail (Jain et al., 2006 and 2007). RelMsm was the only known RSH enzyme present in M. smegmatis, and consequently, a strain of M. smegmatis deleted for the relMsm gene (ΔrelMsm) (Mathew et al., 2004), was expected to show a null phenotype for (p)ppGpp production. In this body of work, we report the surprising observation that the M. smegmatis ΔrelMsm strain is capable of synthesizing (p)ppGpp in vivo. This unexpected turn of events led us to the discovery of a second (p)ppGpp synthetase in this bacterium. The novel protein was found to possess two functional domains – an RNase HII domain at the amino-terminus, and a (p)ppGpp synthetase or RSD domain at the carboxy-terminus. We have therefore named this protein ‘MS_RHII-RSD’, indicating the two activities present and identifying the organism from which it is isolated. Orthologs of this novel SAS protein occur in other species of mycobacteria, both pathogenic and non-pathogenic. In this study, we report the cloning, purification and in-depth functional characterization of MS_RHII-RSD, and speculate on its in vivo role in M. smegmatis.
Chapter 1 reviews the available literature in the field of stringent response research and lays the background to this study. A historical perspective is provided,
starting with the discovery of the stringent response in bacteria in the early 1960s, highlighting the development in this area till date. The roles played by the long and short RSH enzymes, ‘Magic Spot’ (p)ppGpp, the RNA polymerase enzyme complex, and a few other RNA and proteins are described, briefly outlining the inferences drawn from recent global gene expression and proteomics studies. The chapter concludes with a description of the motivation behind, and the scope of the present study.
Chapter 2 discusses the in vivo and in silico identification of MS_RHII-RSD in M. smegmatis. Experiments performed for the genotypic and phenotypic revalidation of M. smegmatis ΔrelMsm strain are described. Detailed bioinformatics analyses are provided for the in silico characterization of MS_RHII-RSD in terms of its domain architecture, in vivo localization, and protein structure prediction. A comprehensive list of the mycobacterial orthologs of MS_RHII-RSD from a few representative species of infectious and non-infectious mycobacteria is included.
Chapter 3 summarizes the materials and methods used in the cloning, purification, and the biophysical and biochemical characterization of full length MS_RHII-RSD and its two domain variants – RHII and RSD, respectively. A detailed description of the purification protocols highlighting the specific modifications and changes made is given. Peptide mass fingerprinting to confirm protein identity, as well as preliminary mass spectrometric, chromatographic, and circular dichroism-based characterization of the proteins under study is also provided.
Chapter 4 deals in detail with the in vivo and in vitro functional characterization of the RNase HII and (p)ppGpp synthesis activities of full length MS_RHII-RSD and its two domain variants - RHII and RSD, respectively. The RNase HII activity is characterized in vivo on the basis of a complementation assay in an E. coli strain deleted for the RNase H genes; while in vitro characterization is done by performing a FRET-based assay to monitor the degradation of a RNA•DNA hybrid substrate in vitro. The (p)ppGpp synthesis activity is characterized in terms of the substrate specificity, magnesium ion utilization, and a detailed analysis of the kinetic parameters involved. A comparison of the (p)ppGpp synthesis activity of MS_RHII-RSD vis-à-vis that of the classical RSH protein, RelMsm, is also provided. Inferences drawn from (p)ppGpp hydrolysis assays and the in vivo expression profile of MS_RHII-RSD in M. smegmatis wild type and ΔrelMsm strains are discussed. Based on the results of these functional assays, a model is proposed suggesting the probable in vivo role played by MS_RHII-RSD in M. smegmatis.
Chapter 5 describes the attempts at generating MS_RHII-RSD overexpression and knockout strains in M. smegmatis, using pJAM2-based mycobacterial expression system, and mycobacteriophage-based specialized transduction strategy, respectively. The detailed methodology and the principle behind the techniques used are explained. The results obtained so far, and the future work and strain characterization to be carried out in this respect are discussed.
Chapter 6 takes a slightly different route and summarizes the work carried out in characterizing the glycopeptidolipids (GPLs) from M. smegmatis biofilm cultures. A general introduction about the mycobacterial cell wall components, with special emphasis on GPLs, is provided. The detailed protocols for chemical composition and chromatographic analyses are mentioned, and the future scope of this work is discussed.
Appendix-1 briefly revisits the preliminary studies performed to determine the pppGpp binding site on M. smegmatis RNA polymerase using a mass spectrometry-based approach. Appendices-2, 3, 4 and 5 give a comprehensive list of the bacterial strains; PCR primers; antibiotics, buffers and media used; and the plasmid and phasmid maps, respectively
Stringent Response in Mycobacteria: Molecular Dissection of Rel
No full textAdaptation to any undesirable change in the environment dictates the survivability of many microorganisms. Such changes generate a quick and suitable response, which guides the physiology of bacteria. Stringent response is one of the mechanisms that can be called a survival strategy under nutritional starvation in bacteria and was first observed in E. coli upon amino acid starvation, when bacteria demonstrated an immediate downshift in the rRNA and tRNA levels (Stent and Brenner 1961). Mutations that rendered bacteria insensitive to amino acid levels were mapped to an ‘RC gene locus’, later termed relA because of the relAxed behavior of the bacteria (Alfoldi et al. 1962). Later on, Cashel and Gallant, showed that two “magic spots” (MSI and MSII) were specifically observed in starved cells when a labeled nucleotide extract of these cells was separated by thin layer chromatography (Cashel and Gallant 1969). These molecules were found to be polyphosphate derivatives of guanosine, ppGpp and pppGpp (Cashel and Kalbacher 1970; Sy and Lipmann 1973), and were shown to be involved in regulating the gene expression in
the bacterial cell, demonstrating a global response, thus fine-tuning the physiology of
the bacterium. Two proteins in E. coli, RelA and SpoT, carry out the synthesis and
hydrolysis of these molecules, respectively, and maintain their levels in the cell
(Cashel et al. 1996; Chatterji and Ojha 2001). On the other hand, Gram-positive
organisms have only one protein Rel carrying out the functions of both RelA and
SpoT (Mechold et al. 1996; Martinez-Costa et al. 1998; Avarbock et al. 1999).
Although Rel or RelA/SpoT has been studied from several systems in detail pertaining to the physiological adaptation, less information is available on the egulation of the protein activity under different conditions. Our studies show that the
RelMsm is composed of several domains (HD, RSD, TGS and ACT) with distinct function. HD and RSD domains, present in the N-terminal half of the protein, harbor catalytic sites for the hydrolysis and the synthesis of (p)ppGpp, respectively. TGS and ACT domains, on the other hand, are present at the C-erminal half of the protein and have regulatory function. It, therefore, appears that a communication exists between these domains, to regulate protein activity. It was shown earlier, while studying Rel from S.equisimilis, that there exists an interaction between the C-terminal and the N-
terminal of the protein which determines the kind of activity (synthesis/hydrolysis),
the protein should demonstrate (Mechold et al. 2002). Later, the N-terminal half
crystal structure of the same protein suggested an inter-domain “cross-talk” between the HD and the RSD domain that controls the synthesis/hydrolysis switch depending on cellular conditions (Hogg et al. 2004).
In the present work, studies have been carried out to understand a Gram-
positive Rel in greater detail and to find out how the opposing activities of Rel are
regulated so that a futile cycle of synthesis and hydrolysis of (p)ppGpp, at the expense of ATP, can be avoided. The work has been divided into several chapters describing
studies on various aspects of the protein.
Chapter 1 outlines the history of the stringent response and summarizes the
information available about the stringent response in various systems including plants.
Several roles that (p)ppGpp plays in different bacteria have been examined. A special mention on the crystal structure of RelSeq has been made with respect to the regulation of activity. Also, the information available regarding the effects of (p)ppGpp on RNA polymerase has been documented. Role of ppGpp in plants has been discussed in great detail with special emphasis on abiotic stresses.
Since different functional domains have been identified in RelMsm, the protein
has been divided into two halves and they have been discussed separately in the form
of two chapters.
Chapter 2 describes the N-terminal half of the Rel protein of M. smegmatis in greater detail. Out of the several domains identified, the role of the two domains
present in the N-terminal half of the protein has been studied. The N-terminal half
shows both synthesis and hydrolysis activities. Importantly, we find that the protein is active even in the absence of accessory factors such as ribosome and uncharged tRNA, unlike RelA of E. coli. Moreover, deletion of the C-terminal half of the protein leads to a much higher synthetic activity, clearly indicating that the C-terminus is involved in regulating the activity of the protein. Both TGS and ACT domains (the two domains found in the C-terminal half of the protein) have been found to play a regulatory role. The results also indicate that all the deleted constructs are active both in vitro and in vivo.
Chapter 3 discusses the C-terminal half of the protein and its role in the
multimerization observed in RelMsm. We show that multimerization of Rel protein is
due to the inter-molecular disulfide cross-linking. Furthermore, we find that the
monomer is the active species in vivo. One of the fascinating points about the C-
terminal half is that it is largely unstructured. Additionally, the C-terminal half cannot complement the N-terminal part of the protein when provided in trans, demonstrating further, the requirement of an intact protein for bringing about regulation of Rel activity. This requirement in cis suggests the presence of an intra-molecular
communication between the N- and the C-termini, as a mediator of protein regulation.
Further, presence of uncharged tRNA increases pppGpp synthesis and down-regulates
its hydrolysis in the wildtype protein. However, the uncharged tRNA-mediated
regulation is absent in the deleted construct with only the N-terminus half, indicating that uncharged tRNA binds to the C-terminal half of the protein. Several cysteine mutants have been constructed to understand their role in the regulation of Rel activity. The results suggest that one cysteine, present at the C-terminus, is required for intra-molecular cross-talk and the uncharged tRNA-mediated regulation.
A detailed characterization of the communication between the two halves of
the protein has been attempted in Chapter 4. Surface plasmon resonance experiments
carried out on the different cysteine mutants discussed in Chapter 3, for uncharged
tRNA binding indicate that all the mutants bind to uncharged tRNA with near-equal
affinities as the wildtype protein. This study suggests that the non-responsiveness for tRNA seen in one of the cysteine mutants is due to the loss of inter-domain
interaction, while the binding of protein to accessory factors is unaffected. Fluorescence resonance energy transfer has been carried out to observe domain
movement in the presence of accessory factors. Distances between the different
domains scattered in this ~90 kDa protein, measured by FRET technique, are suggestive of an inter-domain cross-talk, specifically between C338 and C692, thereby regulating the activity of this enzyme. We show, for the first time, that the product of this protein, (p)ppGpp can bind to the C-terminal half making it unstructured, and can, therefore, regulate the protein activity.
Chapter 5 is an effort to characterize the promoter of rel from M. tuberculosis. This study was undertaken in order to develop an expression system in mycobacteria. The +1 transcription and the translation start sites have been identified. The –10
hexamer for the RNA polymerase binding has also been mapped using site-directed
mutagenesis and is found to be TATCCT. This promoter is also unusually close to the +1 transcription start site. The promoter is specific for mycobacteria and does not
function in E. coli. Additionally, the promoter is found to be constitutive in M.
smegmatis; however, the possibility of it being regulated in M. tuberculosis cannot be
ruled out.
Appendix section discusses, in short, the phylogenetic analysis of the mycobacterial Rel sequences. Diagrams of the plasmids used in this study have been provided. Mass spectra recorded for the in vitro synthesized and purified pppGpp and
the trypsin digest of the full-length Rel protein have also been given.
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Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ
No full textMycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the ω subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc2155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting
Role of RNA Polymerase ω Subunit in Metabolism and Stress Response
No full textRNA polymerase (RNAP) is the key enzyme in transcription and it is a multi-subunit enzyme made up of alpha, beta, beta prime and omega subunit in the stoichiometry of α2ββω. Except for the smallest subunit ω (rpoZ), all the subunits are essential for the cell survival. No clear phenotype was observed for the ΔrpoZ strain of E. coli for many years. Recently we isolated several dominant negtaive mutants of ω. These ω mutants were found to be structured as compared to the native ω which is unstructured. Mutant RNAP with the structured ω was found to be defective at the initiation step of transcription. This study showed the structural importance of ω subunit. Also, ω is linked to stringent response and its role is associated with key players of the stringent response i.e. ppGpp and protein DksA. ppGpp and DksA have been extensively studied with respect to the role played by them in cell survival under the stress. DksA and ppGpp show a more pronounced effect in vivo as compared to that of in vitro. ω has been found to be involved in binding of sigma factors and ppGpp to RNAP and its role has been evaluated in the present study in a more detailed manner. Our studies revealed both the structural and functional role of ω. The functional role of ω in stress response and its role in the distribution of RNAP across the E. coli genome has been studied. The importance of the unstructured ω in maintaining the catalytic activity of RNAP has been analysed. Also, the importance of flexible ω in ppGpp and σ factors binding to RNAP has been deciphered.
Chapter 1 gives a brief introduction about the functional modulation of RNA polymerase. Transcription modulators which interact with RNA polymerase to orchestrate the transcription of genes are discussed.
Chapter 2 presents our findings on the functional and structural role of ω subunit in the interaction of ppGpp to RNAP and its physiological importance in E. coli.
Chapter 3 documents the assembly of the wild type ω and its dominant negative variant, ω6 with reconstituted RNAP (core1: α2ββ′). Subsequently, the interaction of σ-factors with reconstituted RNAP (core2: α2ββ′ω; mutated core2: α2ββ′ω6) has been described.
Chapter 4 provides a broader perspective of the role played by ω in transcriptional machinery by looking into the gene selection pattern of ω-less RNA polymerase. Growth phenotype of ω deleted strain with various carbon substrates and its tolerance to different environmental stress like osmotic stress, pH and antibiotic, using phenotype microarray has been examined.
Chapter 5 summarises the work that has been documented in this thesis.
Appendix-
Chapter 6 describes the co-immunoprecipitation studies which were done to analyse the binding profile of RNA polymerase in ΔrpoZ and ΔdksA strains. Phenotypic microarray and promoter activity assay were done to analyse the correlation of these factors in vivo.
Appendix-
Chapter 7 describes the differential role played by ω subunit in Gram-positive and Gram-negative bacteria
Role of an RNA Polymerase Interacting Protein, MsRbpA, from Mycobacterium smegmatis in Phenotypic Tolerance to Rifampicin
No full textRifampicin is a non-competitive inhibitor of bacterial RNA polymerase (RNAP). The knowledge about the mechanism of action of this drug has emanated from the genetic and the biochemical studies carried out on Escherichia coli RNAP. The complete picture about the steric mechanism was obtained from structural studies on Thermus aquaticus core RNAP in complex with rifampicin. Resistance to rifampicin has been attributed to mutations in its binding pocket lying within the β-subunit of RNAP. The phenomenon of molecular tolerance to this drug came to light with the discovery of differential inhibition of transcription from σ70- and σ32- dependent promoters by rifampicin. After the discovery of the differential inhibition of transcription from σ70- and σ32- dependent promoters by rifampicin, similar instances of proteins were reported. In all such cases, their association with RNAP reduced its susceptibility to rifampicin. In an independent line of study, a new protein, RbpA, was discovered in Streptomyces coelicolor. This protein has the capability of interacting with RNAP, causes rifampicin tolerance to RNAP activity in vitro and leads to basal levels of rifampicin resistance in vivo. Moreover, this protein has sequence homologs exclusively in the actinomycete family, with nearest neighbors in mycobacteria. Interestingly, when rbpA null mutants in S. coelicolor were transformed with M. tuberculosis rbpA gene, the resistance to rifampicin grew from 0.75μg/ml to 2μg/ml in vivo, which pointed towards an analogous role of rbpAMTB. Saturation mutagenesis studies carried out on M. tuberculosis have placed rbpAMTB (Rv2050) on the list of genes imperative for optimal growth. The similarities and the speculations over mycobacterial RbpA made a compelling case in its favour for deciphering the role it played in the mycobacterial paradigm especially in the backdrop of rifampicin tolerance. This work focuses on the discovery of MsRbpA in M. smegmatis as an RNAP-interacting protein, characterization of MsRbpA with respect to its role in conferring phenotypic tolerance to rifampicin, molecular mechanism of the release of rifampicin from RNAP-rifampicin complex, location of MsRbpA on RNAP and studies on the probable role of MsRbpA in a rifampicin-resistant strain.
The thesis is organized as follows:
Chapter 1 deals with the literature survey on rifampicin with an all-inclusive perspective. It provides a brief history on the evolution of antibiotics as principal inhibitors against essential macromolecular assemblies, like RNAP and ribosomes, which control major cellular processes. Subsequently, we discuss the inhibitors of the transcription process, with a major emphasis on the mechanisms of inhibition of transcription activity by rifampicin. We also present an in-depth bibliomic analysis of the response to rifampicin across the microbial spectrum, which scratches the physiological and molecular landscape in a comprehensive manner. Finally, we present the justification for undertaking this work, and the proximate aims of this study.
Chapter 2 informs about the identification of MSMEG_3858 as an RNAP-interacting protein. It details the cloning, expression and purification of MsRbpA. The focus then shifts towards the interact omics of MsRbpA and RNAP. Studies on the in vivo expression of MsRbpA form the concluding part of this chapter.
Investigation into the outcome of interaction between MsRbpA and RNAP forms the central theme of
Chapter 3. In this regard, first of all, we report the in vitro reconstitution of a heterologous mycobacterial core RNAP and its competence in carrying out gel-based in vitro promoter-specific transcription assays. Consequently, we apply this transcription apparatus in judging the role of MsRbpA in preventing the rifampicin-mediated inhibition of transcription activity, both in single- and multiple-round gel-based transcription assays. As a corroboration of this work, it is shown as to how the induction of MsRbpA in vivo causes an increase in the rifampicin-tolerance levels of M. smegmatis. Finally, we probe the existence of any possible interaction between MsRbpA and rifampicin as a prelude to ensuing investigations into the mechanism of phenotypic tolerance to rifampicin.
In Chapter 4 we decipher the molecular mechanism of MsRbpA-mediated release of rifampicin from RNAP-rifampicin complex. A two-pronged strategy comprising of fluorescence-based and mass spectrometry-based approaches helps us to elucidate the aforesaid mechanism in greater detail. As a result, the location of interaction of MsRbpA on M. smegmatis RNAP is found to be at the junction of β and β' subunit.
Chapter 5 attempts to explain the existence of RbpA homologs in microorganisms with two copies of β-subunit, one of which is Rifampicin susceptible (RifS) and the other being Rifampicin resistant (RifR). Using M. smegmatis as a model organism, we induce rifampicin resistance in M. smegmatis and create 3 variants with graded rifampicin resistance. After confirming the non-revertant nature of these strains, we assay the role of MsRbpA in rescuing the activity of RifR RNAPs at their respective IC50 values. The results show a redundancy of MsRbpA in rescuing RifR RNAPs. Alongside, we compare the colony morphology of MsRbpA overexpressing strain and the RifR strains at subinhibitory concentrations of rifampicin. The comparison shows a similarity in colony morphology typical to stress-induced conditions. Combining these results, we attempt to visualize the role of MsRbpA in species carrying two copies of the β-subunit.
Chapter 6 studies the coevolution of RbpA and RNAP in the actinomycete phylum. The studies presented involve bioinformatics and statistics to decipher the cause of the exclusive existence of this protein in actinobacteria. The results show that RbpA appears to have co-evolved with actinobacterial RNAP as a defense system. This could be one of the ways of survival in the soil environment where metabolites like rifampicin are secreted by the producing organisms during struggle for existence.
Chapter 7 summarizes the work presented in this thesis
Probing Macromolecular Reactions At Reduced Dimensionality : Mapping Of Sequence Specific And Non-Specific Protein-Ligand lnteractions
No full textDuring the past decade the effects of macromolecular crowding on reaction pathways is gaining in prominence. The stress is to move out of the realms of ideal solution studies and make conceptual modifications that consider non-ideality as a variable in our calculations. In recent years it has been shown that molecular crowding exerts significant effects on all in vivo processes, from DNA conformational changes, protein folding to DNA-protein interactions, enzyme pathways and signalling pathways. Both thermodynamic as well as kinetic parameters vary by orders of magnitude in uncrowded buffer system as compared to those in the crowded cellular milieu. Ignoring these differences will restrict our knowledge of biology to a “model system” with few practical understandings. The recent expansion of the genome database has stimulated a study on numerous previously unknown proteins. This has whetted our thirst to model the cellular determinants in a more comprehensive manner. Intracellular extract would have been the ideal solution to re-create the cellular environment. However, studies conducted in this solution will be contaminated by interference with other biologically active molecule and relevant statistical data cannot be extracted out from it. Recent advances in methodologies to mimic the cellular crowding include use of inert macromolecules to reduce the volume occupancy of target molecules and the use of immobilization techniques to increase the surface density of molecules in a small volumetric region. The use of crowding agents often results in non-specific interaction and side-reactions like aggregation of the target molecules with the crowding agents themselves. Immobilization of one of the interacting partners reduces the probability of aggregation and precipitation of bio-macromolecules by restricting their degrees of freedom. Covalent linkage of molecules on solid support is used extensively in research for creating a homogeneous surface of bound molecules which can be interrogated for their reactivity. However, when it comes to biomolecules, direct immobilization on solid support or use of organic linkers often results in denaturation. The use of bio-affinity immobilization techniques can help us overcome this problem. Since mild conditions are needed to regenerate such a surface, it finds universal applicability as bio-memory chips. This thesis focuses on our attempts to design a physiologically viable immobilization technique for following rotein-protein/protein-DNA interactions. The work explores the mechanism for biological interactions related to transcription process in E. coli.
Chapter 1 deals with the literary survey of the importance and effects of molecular crowding on biological reactions. It gives a brief history of the efforts been made so far by experimentalists, to mimic macromolecular crowding and the methods applied. The chapter tries to project an all-round perspective of the pros and cons of different immobilization techniques as a means to achieve a high surface density of molecules and the advancements so far.
Chapter 2 deals with the detailed technicality and applicability of the Langmuir-Blodgett method. It discusses the rationale behind our developing this technique as an alternate means of bio-affinity immobilization, under physiologically compatible conditions. It then goes on to describe our efforts to follow the sequence-specific and sequential assembly process of a functional RNA polymerase enzyme with one immobilized partner and also explore the role of omega subunit of RNAP in the reconstitution pathway. This chapter uses the assembly process of a multi-subunit enzyme to evaluate the efficiency of the LB system as a universal two-dimensional scaffold to follow sequence-specific protein-ligand interaction.
Chapter 3 discusses the application of LB technique to quantitatively evaluate the kinetics and thermodynamics of promoter-RNA polymerase interaction under conditions of reduced dimensionality. Here, we follow the interaction of T7A1 phage promoter with Escherichia coli RNA polymerase using our Langmuir-Blodgett technique. The changes in mechanistic pathway and trapping of kinetic intermediates are discussed in detail due to the imposed restriction in the degrees of freedom of the system. The sensitivity of this detection method is compared vis-a-vis conventional immobilization methods like SPR. This chapter firmly establishes the universal application of LB technique as a means to emulate molecular crowding and as a sensitive assay for studying the effects of such crowding on vital biological reaction pathway.
Chapter 4 describes the mechanistic pathway for the physical binding of MsDps1 protein with long dsDNA in order to physically protect DNA during oxidative stress. The chapter describes in detail the mechanism of physical sequestering of non-specific DNA strands and compaction of the genome under conditions where a kinetic bottleneck has been applied. The data obtained is compared with results obtained in the previous chapter for the sequence-specific DNA-protein interaction in order to understand the difference in recognition process between regulatory and structural proteins binding to DNA.
Chapter 5 deals with the evaluation of the σ-competition model in E. coli for three different sigma factors (all belonging to the σ-70 family). Here again, we have evaluated the kinetic and thermodynamic parameters governing the binding of core RNAP with its different sigma factors (σ70, σ32and σ38) and performed a comparative study for the binding of each sigma factor to its core using two different non-homogeneous immobilization techniques. The data has been analyzed globally to resolve the discrepancies associated with establishing the relative affinity of the different sigma factors for the same core RNA polymerase under physiological conditions.
Chapter 6 summarizes the work presented in this thesis. In the Appendix section we have followed the unzipping of promoter DNA sequence using Optical Tweezers in an attempt to follow the temporal fluctuations occurring in biological reactions in real time and at a single molecule level
(p)ppGpp and c-di-GMP : A Tale of Two Second Messengers in Mycobacterium smegmatis
No full textNucleotide based second messengers are known to regulate wide variety of processes in all domains of life. Two such bacterial second messengers are (p)ppGpp (guanosine tetra- or pentaphosphate) and c-di-GMP (cyclic dimeric guanosine monophosphate). The alarmone (p)ppGpp is synthesized by bacteria to face any kind of stress; while the signalling nucleotide c-di-GMP is synthesized principally to switch from motile (planktonic) to sessile (biofilm) life style. Apart from mediating the said functions, these nucleotides also regulate transcription, translation, replication, virulence and pathogenicity of the several bacterial species. In this work, we have tried to uncover novel functions or phenotypes that are governed by the second messengers (p)ppGpp and c-di-GMP in Mycobacterium smegmatis. In M. smegmatis, (p)ppGpp and c-di-GMP are synthesized and degraded by the bifunctional proteins RelMsm and DcpA, respectively. The architecture of both the proteins is similar; the synthesis and hydrolysis domains for the second messengers occur in tandem. The knockout mutants of relMsm and dcpA genes, ∆relMsm and ∆dcpA, have been used in this study to uncover the novel functions of these second messengers in mycobacterial physiology. Chapter 1 provides is an overview of the current literature pertaining to (p)ppGpp and c-di-GMP. An historical perspective with regard to the discovery of the (p)ppGpp and c-di-GMP is given. The metabolism of these second messengers has been discussed. This has been followed by the description of various functions governed by the second messengers. Finally, the scope of the current work has been outlined.
Chapter 2 investigates the effect of disrupting (p)ppGpp and c-di-GMP signalling on the antibiotic sensitivity in M. smegmatis. Using Phenotype Microarray (PM) technology, the growth of ∆relMsm and ∆dcpA knock out strains was compared to those of the wild-type and respective complemented strains in 240 different antimicrobials. It was found that the knockout mutants displayed enhanced survival in the presence of multiple antibiotics. The PM data was corroborated by the independent determination of minimum inhibitory concentrations of seven different antibiotics. Finally, the plausible reasons for the multidrug resistance of ∆relMsm and ∆dcpA strains have been discussed.
Chapter 3 explores how the impairment of (p)ppGpp and c-di-GMP alters the cell wall of M. smegmatis. Thin layer chromatography analysis of cell wall fractions such as glycopeptidolipids (GPLs), mycolic acids, polar and apolar lipids was carried out. It was found that the amount of GPLs and polar lipids were reduced in the ∆relMsm and ∆dcpA knockout strains.
Chapter 4 explores the effect of (p)ppGpp and c-di-GMP on the growth, cell morphology and cell division in M. smegmatis. It was found that the ∆relMsm and ∆dcpA knockout strains have slow growth compared to those of the wild type and respective complemented strain. The overproduction of (p)ppGpp and c-di-GMP, achieved through overexpression of Rel and DcpA proteins, encased the overexpression strains relOE and dcpAOE in a biofilm like matrix. The higher levels of (p)ppGpp and c-di-GMP caused M. smegmatis assume coccoid morphology. Microscopy analyses revealed that the ∆relMsm and ∆dcpA strains are elongated, multinucleate and multiseptate.
Chapter 5 explores effects of (p)ppGpp and c-di-GMP on the global gene expression profile in M. smegmatis. Many genes were shown to be differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Genes regulating cell division, cell wall biosynthesis, superoxide metabolism or reactive oxygen species metabolism and genes encoding transporters were differentially expressed in the ∆relMsm and ∆dcpA knockout mutants. The microarray data were corroborated by quantitative real-time PCR. Gene expression data explained the multidrug resistance, the reduction in the level of GPLs and polar lipids, slow growth, changes in cell morphology and defective cell division exhibited by the ∆relMsm and ∆dcpA knockout mutants.
Chapter 6 summarizes the entire work embodied in the thesis.
Appendix 1 lists the 240 antimicrobials compounds and their mode of action for which antibiotic sensitivity of the ∆relMsm and ∆dcpA knockout mutants was tested.
Appendix 2 lists the growth differences among the knockout, wild type and complemented strains in the form of area under curve values.
Appendix 3 lists the genes that were differentially expressed in the ∆relMsm and ∆dcpA knockout strains.
Appendix 4 is a comprehensive review on the kinetic and thermodynamic parameters governing the sigma factor competition in Escherichia coli and how (p)ppGpp and anti-sigma factors regulate this competition among sigma factors for the limited pool of core RNA polymerase in E. coli
Newer Insights On Structure, Function And Regulation Of Dps Protein From Mycobacterium smegmatis
No full textThe first chapter will provide an introduction to the physiology, pathogenesis and biology of mycobacteria. Host-pathogen interactions, different modes of resistance of the bacteria, adaptations for survival under nutrient and oxygen depleted conditions has been discussed. This is followed by a general discussion on gene expression and regulation in the microbe. The physiology of bacteria under stresses from the view of the transcriptional regulation of specific genes has also been discussed. The scope and objective of the present study in M. smegmatis covered in the thesis has been considered at the end. The next chapter discusses the characterization of msdps promoter in vivo with the help of reporter gene assay technologies. With the advent of promoterless E. coli-mycobacterium shuttle vectors, activity assays can be easily performed to characterize unknown upstream putative promoter sequences of genes. Both the 1 kb upstream as well as a 200bp upstream region of msdps gene has been characterized by. Primer extension analysis and subsequent site directed mutagenesis studies reveal +1 transcription start site and the promoter consensus sequence for the msdps gene respectively. Next chapter comprises of the method of constructing heterologous in vitro transcription machinery in mycobacteria. It is followed by characterization of transcription initiation at two dps promoters of M. smegmatis. A novel pull-down assay has been designed which enabled us to identify the sigma factors in the reconstituted RNA polymerases to be associated with the respective dps promoters and to compare the regulation of the two genes at transcription level. Further characterization through single round in vitro transcription at mycobacterial promoters has been attempted. The following two chapters provide some newer insights into the structure-function relationship of the first Dps molecule, MsDps (MsDps1) with respect to its DNA binding activity. The DNA binding activity is associated with the higher oligomeric form only. With the help of time resolved anisotropy and Förster Resonance Energy Transfer (FRET) experiments, we have monitored the nature of Dps dodecamer-DNA complex and mapped the distance between the N and C169 position in the absence and the presence of DNA. A new computational programme, Maximum Entropy Method (MEM) has been applied successfully to analyze data obtained from phase-modulation (Phi-M) lifetime experiments in order to get distribution of lifetime. In the last chapter a new method is adopted to predict amino acids important for stabilizing the interface in a trimeric structure. Subsequently, single and double amino acid mutants of the native MsDps protein has been constructed through site directed mutagenesis and are scored for the ability of the mutants to oligomerize under conditions similar to that of the native protein. This helped us to propose a hypothetical model of the overall mechanism of the protein oligomerization process in solution
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